Worldwide, gastrointestinal infections and diarrheal diseases result in significant morbidity and mortality with approximately two million deaths each year. However, current diagnostic techniques fail to determine a causal infectious agent in 30-50 percent of cases. Astroviruses (AstV) are known to cause acute gastroenteritis in humans, other mammals and avian hosts. All eight genotypes of AstV known to infect humans are phylogenetically closely related and represent a single AstV species within the Astroviridae family. Using a combination of high throughput sequencing and degenerate PCR, we detected sequences of twelve highly divergent AstV in human stool samples from different continents. Comparative analysis of complete and partial genome sequences suggest that these highly divergent AstV variants can be classified into four novel AstV species, all of which are genetically closer to animal AstV species than to the previously known human AstV species. To prevent further spread of these novel viruses and their disease in humans worldwide, it's crucial to determine their capability of infecting humans, genetic diversity, prevalence and disease association. Data generated will help design sensitive molecular reagents and provide groundwork for future research on these novel viruses. Our hypothesis is that these four novel AstV species include several divergent genotypes which are capable of infecting humans and are associated with gastroenteritis. We propose two specific aims necessary to achieve our long term goal of understanding and reducing human disease burden caused by these novel viruses. Specific aim 1 is to characterize the full range of viral genetic diversity, develop accurate diagnostic assays and determine disease association. We have accumulated a large collection of clinical and field specimens from Mexico, Unites States, Nepal, Pakistan, India, Nigeria, Tunisia and France, that will allow us to perform these studies. Since AstV can only be propagated in cell lines derived from their natural host, specific aim 2 is to define the host tropism of these novel viruses using cell culture and to develop serological assays. Cell lines from several mammalian species will be used for infection assays and to make pure virus strains. For serology, virus neutralization and luciferase immunoprecipitation system (LIPS) assays will be optimized to detect and measure anti-virus antibodies in human serum samples thereby confirming or refuting them as human viruses. The same assays will also be used to determine viral seroprevalence, because the latter will indicate the proportion of the human populations susceptible or immune to infection by these viruses and their diseases. The proposed molecular and biological characterization of these novel viruses is critical to understand their pathogenic potential and to develop methods to prevent or interrupt their transmission. PUBLIC HEALTH RELEVANCE: Molecular characterization of the novel viruses associated with acute gastroenteritis is crucial to develop sensitive and accurate diagnostic assays. Seroprevalence studies will determine the proportion of the human populations susceptible to diseases caused by these novel astroviruses. Results of this study will help to formulate strategies to interrupt transmission of these new viruses and their diseases in humans.